BACKGROUND: Our goal was the efficient labelling of highly purified human gammaglobulin. This radioactive protein fraction can be used as a basic compound of radiopharmaceutical formulation for inflammation lesion diagnosis. This application was experimentally illustrated in animal models with artificially induced inflammatory lesions after turpentine oil injection into mouse leg muscle.
MATERIALS AND METHODS: Hydrazine nicotinamine derivative of human gammaglobulin (IgG-HYNIC) was synthesized according to Abrams method. The radionuclide: technetium ^99mTc has been introduced into protein molecules by indirect method incorporation in phosphate buffer, pH 7.4, in the presence of stannous chloride as a reducing agent for sodium pertechnetate, and EDTA as a coligand. Radiochemical purity was estimated by thin layer chromatography. The stability of labelled IgG-HYNIC derivative in human serum in presence of copper, cobalt, iron and manganum salts was analyzed by HPLC method (BioSEP SEC 4000, eluent: 0.1mol/L phosphate). Inflammation lesions were induced in Balb/3 mice muscles by injection of 0.2 ml turpentine oil into the leg muscle. Five days later, inflammation lesions were visualized by hIgG-HYNIC- ^99m Tc injections. The tracer accumulation in tissue was evaluated by gamma camera at 1 to 24 hour intervals.
RESULTS: Efficiency of technetium99m Tc human gammaglobulin labelling (pH 7.4, temp. 37°C) was strictly dependant on ligand and coligand presence in the reaction mixture. Labelling of IgG molecules without any supplements resulted in very low efficiency, never exceeding the range of 5%. Presence of EDTA or hydrazine nicotinamide (HYNIC) conjugated with IgG increased radiolabelling efficiency to 50%. IgG-HYNIC derivative in EDTA presence enables us to reach value above 95% radiochemical purity. Stability of IgG-HYNIC derivative labelled with technetium ^99m Tc decreased rapidly in serum in time - up to 70% of initial value in 30 minutes and only 20% during further 4 hr incubation. This means that as much as 80% of radiotracer present in IgG molecules has been dissociated during incubation with serum. This forced us to find proper conditions for improving the stability of radioactive IgG-HYNIC conjugate in circulating serum for at least six hours. It was achieved by using a reaction medium supplement with divalent metal cations in the following compounds: MgCl₂, CoSO₄, Cu (NO₃)₂ and FeCl₂ in equimolar ratio to EDTA. Scintigraphy of ^99mTc gammaglobulin in artificially induced inflammatory lesions of mouse thigh muscle showed a 4 times higher accumulation of the tracer after 6 hours post injection, and 6 times higher after 24 hours.
CONCLUSIONS: A human gammaglobulin derivative (hIgG-HYNIC) labelled with technetium ^99mTc by indirect method with high radiochemical purity can be a basic compound of formulation for infection/inflammation scintigraphy.